The Association And Mechanism Between Gut Microbiota Metabolism Ursodeoxycholic Acid And Ischemic Stroke

Background and objectiveIschemic stroke has been demonstrated to cause gut microbiota dysbiosis in which bile acids(BA)metabolism might be involved.The aim of the study was to find the connection of gut microbiota to BA metabolism disturbance in ischemic stroke and check the effects of targeting BA metabolism for ischemic stroke therapy such as ursodeoxycholic acid(UDCA),in alleviating neuroinflammation through TGR5NLRP3 signaling pathways.Methods1.Blood samples of 56 subjects and mRS at 3 months after discharge from Nanfang Hospital were collected,including healthy patients(n=28)and acute stroke patients(n=28).Serum bile acids were detected by mass spectrometry,the changes of bile acids and biochemical index between two groups were observed and the correlation between UDCA and outcome were compared.2.The BA profiles in liver,intestine,brain and serum of mice with ischemic stroke induced by transient middle cerebral artery occlusion(tMCAO)were detected by ultraperformance liquid chromatography-tandem mass spectrometry and the composition ofgut microbiota were examined by 16S rRNA gene sequencing.Spearman method was used to analyze the correlation between gut microbiota and bile acid changes of intestine and serum after stroke mouse.3.It was observed that UDCA is the common change of bile acids between stroke mouse and patients,and whether UDCA intervention has a protective effect after stroke.Infarct volumes and short-term neurologic scores were evaluated by TTC and mNSS respectively.The long-term neurologic scores were evaluated by Morrie’s water maze(MWM).The status of microglial polarization was performed by immunofluorescence staining.The morphological changes and apoptosis of neurons were observed by Nissl and Tunel staining.In order to clarify the mechanism;In vitro experiments,LPS was used to stimulate mouse microglia BV-2 cells to produce inflammatory factors.After UDCA intervention,the expression of TGR5 receptor and its inflammatory factors was observed by qPCR,and then the expression of TGR5 was inhibited by siRNA.the change of NLRP3 inflammasome expression was observed after UDCA intervention;In vivo experiments,to elucidate the mechanism of the effect of UDCA on neuroprotection,TGR5 knockout AAV9-CRISPR and PKA inhibitor H89 were administered intracerebroventricularly and intraperitoneally at 21d and 1h before MCAO.The expression of TGR5\cAMP\PKA\NLRP3 inflammation was detected by western blot.Results1.HDL in stroke patients were lower than the normal subjects,and creatinine were significantly higher than the normal group(P<0.05).Metabonomics analysis showed that the content of secondary bile acid was decreased in stroke patients,especially the UDCA content was significantly decreased(P<0.05).It was found that the content of UDCA in bad outcome(45.20±8.08)was lower than that in good outcome(272.67±72.48)(P<0.05).UDCA was a protective factor for poor prognosis(OR,0.89;95%CI:0.72-0.96;p=0.032).2.MCAO mouse showed decreased neurological function and the area of cerebral infarction.Compared with the normal group,there were a significant change in gut microbiota in stroke mouse,the results showed that the diversity of gut microbiota decreased.We found that Bacteroidetes was enriched and Firmicutes was decreased.According to bile acid metabolomics results,it was found that bile acid among plasma,intestinal and brain were decreased,especially secondary bile acid UDCA(P<0.05).3.UDCA could reduce the infarction size of brain and improve the functi on of neural and cognitive ability.Further studies found that UDCA could reduce M1 microglia and neuronal degeneration in the hippocampus,increased expression of TGR5 and PKA protein,and decreased expression of NLRP3 and inflammatory factor IL-1β.In vitro experiments,UDCA interfered with LPS-sti mulated BV-2 cells,and it was found that the increasing expression of TGR5 and the decreasing expression of NLRP3 inflammasome.After the intervention of siRNA-TGR5,the anti-inflammatory effect of UDCA has been disappeared.In vivo:After knocking out TGR5 by AAV9-CRISPR and PKA inhibitor H89,the protective effect of UDCA also has been disappeared,the area of cerebral infarction increased and the release of inflammatory factors increased.ConclusionsThere was significant change in UDCA in stroke patients and mouse,we found that the gut microbiota disorder in mouse after stroke,and UDCA can interfere with the expression of NLRP3 inflammasome through TGR5/cAMP/PKA pathway,reducing neuroinflammation and cerebral infarction area,improving neurological function and cognitive function.